Visualizing and quantifying oxidized protein thiols in tissue sections: a comparison of dystrophic mdx and normal skeletal mouse muscles

Free Radic Biol Med. 2013 Dec;65:1408-1416. doi: 10.1016/j.freeradbiomed.2013.09.024. Epub 2013 Oct 3.

Abstract

Reactive oxygen species (ROS) are not only a cause of oxidative stress in a range of disease conditions but are also important regulators of physiological pathways in vivo. One mechanism whereby ROS can regulate cell function is by modification of proteins through the reversible oxidation of their thiol groups. An experimental challenge has been the relative lack of techniques to probe the biological significance of protein thiol oxidation in complex multicellular tissues and organs. We have developed a sensitive and quantitative fluorescence labeling technique to detect and localize protein thiol oxidation in histological tissue sections. In our technique, reduced and oxidized protein thiols are visualized and quantified on two consecutive tissue sections and the extent of protein thiol oxidation is expressed as a percentage of total protein thiols (reduced plus oxidized). We tested the application of this new technique using muscles of dystrophic (mdx) and wild-type C57Bl/10Scsn (C57) mice. In mdx myofibers, protein thiols were consistently more oxidized (19 ± 3%) compared with healthy myofibers (10 ± 1%) in C57 mice. A striking observation was the localization of intensive protein thiol oxidation (70 ± 9%) within myofibers associated with necrotic damage. Oxidative stress is an area of active investigation in many fields of research, and this technique provides a useful tool for locating and further understanding protein thiol oxidation in normal, damaged, and diseased tissues.

Keywords: A.U.; BODIPY FL–N-(2-aminoethyl)maleimide; C57; C57Bl/10Scsn; FLm; Free radicals; H&E; Histochemistry; Murine; Muscular dystrophy; N-ethylmaleimide; NEM; Oxidative stress; PBS; PFA; PVA; Protein thiol; ROS; Reactive oxygen species; SDS; Skeletal muscle; Sulfhydryl; TCA; TCEP; arbitrary units; hematoxylin and eosin; paraformaldehyde; phosphate-buffered saline; polyvinyl acetate; reactive oxygen species; sodium dodecyl sulfate; trichloroacetic acid; tris(2-carboxyethyl)phosphine.

MeSH terms

  • Animals
  • Dystrophin / analysis*
  • Fluorescent Dyes
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Muscle, Skeletal / cytology*
  • Muscular Dystrophy, Animal
  • Oxidation-Reduction
  • Oxidative Stress
  • Reactive Oxygen Species
  • Staining and Labeling / methods*
  • Sulfhydryl Compounds / chemistry*

Substances

  • Dystrophin
  • Fluorescent Dyes
  • Reactive Oxygen Species
  • Sulfhydryl Compounds